Dankort Lab pLEG/pREG Modular Viral Vector System
The LEG (Lentiviral Expression Gateway) is system a flexible vector system to allow the rapid production of lentiviral and retroviral (REG) vectors that express cDNAs with or without inhibitory RNAs (shRNAmirs) using Gateway technology.
This modular system consists of: a vector backbone (module 1), a cDNA contained in a standard pEntry vector (module 2), genetic markers or fluorescent proteins are encoded downstream of internal ribosome entry sequences and are flanked by attR2 and attL3 sites contained in a pBEG vector (module 3), and, an optional miR30-embedded shRNA flanked by attR3 and attL4 sites (module 4). These modules are combined using a multisite Gateway LR reaction with a destination vector. Destination vectors can be either a pLEX-based lentiviral vector (pLEG R1-R3 or pLEG R1-R4) or a pQCxix-based retroviral expression vector (pREG R1-R3) or pREG R1-R4). Each vector contains a self-inactivating 3'LTR.
The configuration allows the cDNA, genetic marker or fluorescent protein and miRNA to be cloned while maintaining precise order, spacing and orientation into either a lentiviral or retroviral destination vector. cDNAs may be maintained in pEntry vectors (AttL1 and AttL2) and the same plasmids used for multiple LR reactions to rapidly produce a large number of viral expression vectors with variations in selectable marker or miRNA expression (conversely the cDNA could be varied) without the need to characterize each plasmid. Novel miRNAs may be generated and triaged efficiently using a pCheck (AttR1 and AttR2) plasmid. To do this cDNA (from pEntry vectors) are cloned into pCheck R1-R2 behind Renilla luciferase by standard Gateway LR reaction and then targeted with a candidate shRNAmir.
| Module 1 | Destination vectors: pLEG second generation lentiviral vectors and pREG retroviral vectors for 3- and 4-plasmid recombination reactions |
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| Module 2 | pENTRY vector contains cDNA |
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| Module 3 | pBEG R2-L3 vectors contain an ires and selectable marker |
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| Module 4 | pBEG R3-miRNA-L4 contains miR-30 based shRNA for specific gene knockdown |
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Expressing cDNA with marker of your choice (example of 3 plasmid recombination). Choose a vector (here a lentiviral vector), provide a cDNA (in an Entry vector), and choose the desired marker in pBEG R2-L3 (here puromycin resistance).The resulting viral vector can be transduced into cells where cDNA expression is encoded on the same bi-cistronic mRNA with the selectable marker.
Viral vector is generated through a Gateway LR recombination reaction. Virus is produced by transient transfection using second generation packaging plasmids and cells are transduced.
Expressing cDNA and one or more shRNAs with marker of your choice (example of 4 plasmid recombination). Choose a vector (here a lentiviral vector), provide a cDNA (in an Entry vector), choose the desired marker in pBEG R2-L3 (here eGFP), and a shRNA cloned into pBEG R3-shRNAmir-L4.The resulting viral vector can be transduced into cells where cDNA expression and shRNA is contained within the same multifunctional mRNA along with the selectable marker.
Viral vector is generated through a Gateway LR recombination reaction. Virus is produced by transient transfection using second generation packaging plasmids and cells are transduced.
Testing shRNA efficacy using dual luciferase reporter.
| The target cDNA contained in a standard pEntry vector is cloned via Gateway LR reaction into pCheck2 Dest (R1-R2) downstream of Renilla luciferase. Two different shRNAs are compared by normalizing for transfection efficiency via Firefly Luciferase and then for specific knockdown by assessing Renilla Luciferase levels. |
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